Down-regulation of HtrA1 activates the epithelial-mesenchymal transition and ATM DNA damage response pathways

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.     

Neuronal profilin isoforms are addressed by different signalling pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.     

Nanostructured biomolecular detectors: pushing performance at the nanoscale

Nanomaterial-based biosensing strategies offer a number of advantages over traditional molecular diagnostic and cellular analysis techniques, including signal amplification, improved sensitivity and speed, and versatile sensing schemes that can be tailored to a desired target. In this article, we highlight a variety of nanomaterial-based sensors, and discuss the advantages of different nanomaterials compositions and probes of different biomolecular classes. Recent advances in the development of optical, electrical, or electrochemical transduction mechanisms are covered, with special regard to breakthroughs in sensitivity. The works reviewed herein emphasize the improvements that nanomaterials offer in the realm of diagnostic assays and make a solid case for further advancement with automation and multiplexing.     

How to narrow down chromosomal breakpoints in small and large derivative chromosomes – a new probe set

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ～10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.     

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca²⁺. Two family members, ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose. We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.     

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.     

Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules

Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function.     

Integrin β4 regulates SPARC protein to promote invasion

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.     

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism''s physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen''s ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.